The present invention relates to an assay for the detection of haptoglobin (Hp) in milk as a means to predict or detect mastitis or subclinical mastitis in for example dairy cattle.
Mastitis is very costly to the dairy industry due to discarded milk, reduced yield, milk of lower value, increased rates of premature culling and occasional mortality (Esslemont and Spincer, 1993) and is recognised as one of the major diseases adversely affecting dairy cow welfare (Menzies, 1995). Strategies for early detection of mastitis are particularly beneficial to the dairy industry in terms of early treatment of cattle thus improving milk production whilst generally maintaining milk quality and health status of the animals.
Detection of mastitis pathogens typically forms a basis of mastitis diagnosis (Hamann et al, 1997). For example, the bacteria Staphyloccus aureus is known to be a cause of clinical and subclinical mastitis in dairy cattle (Erskine et al, 1987).
GB98/03132 for example describes an immunological assay using a Staphylococcus aureus antigen mixed with a sample of blood taken from dairy cattle to induce a proliferative response in the blood cells.
Additionally measurement of a level of cell proliferation as a means of detecting mastitis may be determined by a number of methods including addition of radioactive nucleotides followed by scintillation counting. However, such tests involve taking a blood sample, which may not be desirable.
Further to this, somatic cell counting (SCC) is often used as a means to detect mastitis where a high somatic cell count is indicative of this condition (Pyörälä and Pyörälä, 1997). However, this method proves to have an undesirable amount of false positive results giving an inaccurate measurement.
Other tests used to detect mastitis include those involved in detecting inflammatory changes. For example, a group of proteins, known as acute phase proteins show a dramatic increase in concentration in blood in response to infection, inflammation or trauma. Such proteins include Haptoglobin (Hp), carbon-reactive protein (CRP) and serum amyloid A (SAA).
WO9522767 discloses an assay for determining CRP in mammals, where the sample is milk. However a later study by Hamann et al, 1997, raised doubt as to whether or not the determination of CRP levels could be used to predict infection, such as mastitis.
Presently, assays for the detection of another acute phase protein, namely haptoglobin, in blood, serum or plasma samples are based on an immunoassay; the ability of Hp to bind to haemoglobin (Hb); or the determination of the peroxidase activity of an haptoglobin/haemoglobin complex.
Hp in human plasma is typically measured by antibody based methods with antiserum specific for human Hp. However, such tests again require blood sampling and more often than not, some form of fractioning of the blood to obtain a plasma sample. This it will be appreciated can be time consuming and requires blood sampling to be carried out.
Original assays based on Hp-Hb binding depended on the finding that formation of the Hp-Hb complex alters the spectrophotomeric absorption characteristic of Hb in proportion to the concentration of Hp in a plasma sample. GB98/03407 discloses an additional method for determining Hp in blood by measuring, using chemical means, the peroxidase activity of an haptoglobin/haemoglobin complex. However, this assay may not be applicable in the measurement of Hp in milk since there is a non-Hp associated peroxidase activity in milk which is likely to affect such an assay.